Nicotianamine synthase: Gene isolation, gene transfer and application for the manipulation of metal assimilation
Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)
vorgelegt der
Mathematisch-Naturwissenschaftlich-Technischen-Fakultät der Martin-Luther-Universität Halle-Wittenberg
von Herrn Dimitar Douchkov geb. 18. November 1971 Sofia, Republik Bulgarien
1. Gutachter: Prof. Dr. Klaus Humbeck 2. Gutachter: Prof. Dr. Nicolaus von Wirèn 3. Gutachter: Prof. Dr. Ulrich Wobus
List of the Abbreviations
A. tumecaciens Agrobacterium tumefaciens Ampr Ampicilin resistance
ATP Adenosine triphosphate
b, bp Base, base pair BSA Bovien serum albumin CaMV Cauliflower mosaic virus
cDNA Complementary DNA
D Dalton
DEPC Diethyl pyrocarbonate
DNA Deoxyribonucleic acid
DTT Dithiotreitol EDTA Ethylendiamine tetraacatic acid
et al. et alii (and others)
g Gram
HEPES N-[2-Hydroxyethyl]-piperazine-N’-[2-ethansulfonic acid] IPTG Isopropyl-ß-D-thiogalactozide
K Kilo L Litre M Molarity m Mili
MOPS N- morpholinopropanesulfonic acid mRNA Messenger RNA
n Nano
OD Optical density
PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction
RACE Rapid amplification of cDNA ends
RNA Ribonucleic acid
SDS Sodium dodecyl sulfate
T-DNA Transferred DNA
Tris Tris-hydroxymethylaminomethane
WT Wild type
TABLE OF CONTENTS
1. INTRODUCTION ... 1
1.1. Iron as a nutrient... 1
1.2. Iron in Plants... 1
1.3. Nicotianamine and Nicotianamine synthase ... 6
2. MATERIALS AND METHODS ... 10
2.1. Materials ... 10
2.1.1. Plant material ... 10
2.1.2. Bacterial strains... 10
2.1.3. Plasmids... 11
2.1.3.1. Vector maps... 11
2.1.4. Primers and oligonucleotides ... 12
2.1.4.1. PCR primers ... 12
2.1.4.2. Sequencing oligonucleotides ... 14
2.1.4.3. Oligonucleotides and DNA probes used for genetical mapping and detection of the barley NAS genes... 15
2.1.5. Enzymes and kits ... 16
2.1.6. Chemicals... 17
2.1.7. Laboratory tools and equipment... 18
2.1.8. Media... 19
2.1.8.1. Bacterial media ... 19
2.1.8.2. Plant growth media ... 20
2.1.9. Software ... 21
2.2. Methods ... 22
2.2.1. Basic cloning methods and sequencing... 22
2.2.2. Extraction of plant genomic DNA ... 22
2.2.3. Extraction of genomic DNA from Methanobacterium thermoautotrophicum... 24
2.2.4. Southern blotting ... 24
2.2.5. Extraction of plant total RNA ... 25
2.2.6. Northern blotting... 26
2.2.7. Radioactive labeling of DNA probes ... 28
2.2.8. DNA-DNA and DNA-RNA hybridization protocol ... 29
2.2.9. Protein overexpression, isolation and purification... 30
2.2.10. Antibody preparation and purification ... 31
2.2.11. Western blot analysis ... 32
2.2.12. Screening of the barley BAC library ... 33
2.2.13. Agrobacterium tumefacience growth and treatment ... 34
2.2.14.1. Arabidopsis thaliana... 35
2.2.14.2. Nicotiana tabacum growth and treatment... 38
2.2.14.3. Lycopersicon esculentum growth and treatment ... 38
2.2.15. Nicotianamine synthase activity assay... 38
2.2.16. Determination of the metal ions concentration... 39
2.2.17. Determination of the Nicotianamine concentration ... 39
2.2.18. Ferric-reductase assay... 39
2.2.19. Chlorophyll content measurement ... 40
3. RESULTS ... 41
3.1. Arabidopsis thaliana wild type experiments ... 41
3.1.1. Expression analysis using DNA arrays with A. thaliana genes... 46
3.2. The NASHOR1 gene ... 48
3.2.1. Expression of the NASHOR1 in E. coli ... 48
3.2.2. NASHOR1 expression in barley... 49
3.2.2.1. The NASHOR1 gene promoter... 50
3.2.2.2. Immunohistochemical localization of NASHOR1... 55
3.2.2.3. Chromosomal localization of barley NAS genes ... 55
3.2.3. NASHOR1 gene transformation in plants ... 57
3.3. Transgenic plants transformed with the 35S:NASARA1 construct ... 61
3.3.1. Cloning of the NASARA1 gene ... 61
3.3.2. NASARA1 transgenic plants ... 61
3.3.3. Physiological experiments with selected T1 lines... 62
3.4. Transgenic plants transformed with the 35S:NASARA5 construct ... 66
3.4.1. Cloning of the NASARA5 gene ... 66
3.4.2. NASARA5 transgenic plants ... 66
3.4.3. Physiological experiments with selected 35S:NASARA5 lines... 67
3.4.4. Enhanced heavy metal tolerance of NASARA5 tobacco ... 73
3.5. Methanobacterium NAS-like gene... 75
3.6. NAS sequence comparison ... 75
3.7. Branched chain α-keto acid dehydrogenase complex (BCKDH) ... 77
4. DISCUSSION... 80
SUMMARY... 91
ZUSAMMENFASSUNG... 93
LITERATURE ... 95
1. Introduction
1.1. Iron as a nutrient
Iron deficiency is the most common nutritional disorder in the world. The numbers are staggering: as many as 4-5 billion people, 66-80% of the world’s population, may be iron deficient; 2 billion people – over 30% of the world’s population – are anemic, mainly due to iron deficiency. Subtler in its manifestations than, for example, protein-energy malnutrition, it presents the heaviest overall toll in terms of ill-health, premature death and lost earnings. Iron deficiency and anemia reduce the work capacity of individuals and entire populations, bringing serious economic consequences and obstacles to national development. Conversely, treatment can raise national productivity levels by 20%. (World Health Organization, 2001).
Iron supplements, iron-rich diets, increasing iron absorption and fortification should be the main tools to combat micronutrient malnutrition. The plants, as the major nutrient source for the people and animals, must be extensively developed and improved. Therefore, basic understanding of iron homeostasis in plants as well as the development of iron efficient crop plants is a major challenge for research and plant breeding.
1.2. Iron in Plants
Iron is a vital micronutrient involved in many cellular processes like electron transport chains in photosynthesis and respiration, enzyme activation, oxygen carrier in nitrogen fixation etc. It is essential for synthesis of the porphyrin ring as a precursor of haeme as well as chlorophyll, haeme synthesis declines under Fe deficiency conditions in the roots. This leads to physiological anoxia in the roots even though oxygen is present (Mori, 1999).
On the other side, excess supply of iron, as well as other essential and non-essential heavy metal ions, is toxic to most species. One of the general symptoms of heavy metal toxicity in higher plants is chlorosis of young leaves.
Often this is associated with low internal Fe concentrations indicating metal-induced inhibition of mobilization, uptake and/or translocation of Fe (Schmidt et al., 1997). Blockage of physiological response of the plants to Fe shortage such as formation of transfer cells, medium acidification and increased Fe-reductase activity by elevated levels of divalent metal cations have been reported (Landsberg, 1982; Alcantara et al., 1994).
Although iron is abundant in most well aerated soils, its availability for plants is limited. In soil, iron is mainly found as stable Fe (III) compounds that are insoluble at neutral pH. Under anaerobic conditions, iron is precipitated as Fe(OH)3 with very low solubility that limits the concentration of ferric irons at pH 7 to about 10–18 M. (Drechsel and Jung, 1998). Decreasing the pH to 4,0 increases the solubility of Fe (III) by a factor of 1000 (M a and Nomoto, 1996). However, the plants demand approximately 10-4 – 10-8 M Fe (III) for normal growth (Mori, 1999). To overcome this shortage the plants have evolved different iron acquisition mechanisms, which can be clearly divided in two groups often designated as Strategy I, and Strategy II (Römheld and Marschner, 1986).
Iron acquisition mechanism of Strategy I plants
All higher plants except Gramineae use the Strategy I mechanism. They facilitate iron uptake by enhanced extrusion of protons leading to acidification of the rhizosphere and thus to solubilisation of iron compounds, and release of reductors or chelators. A root-specific plasma membrane-bound reductase reduces Fe (III) to Fe (II) and the ferrous iron is taken up by root cells through Fe(II) specific membrane sites.
Enhanced Fe (III) reduction activity is the most typical feature of Strategy I. Two closely related ferric-chelate reductases genes (FRO1 and FRO2) of A. thaliana were isolated (Robinson, 1999). They show moderate similarity to yeast ferric-chelate reductases such as FRE1 and FRP1, and to human phagocytic NADPH oxidase gp91phox. These enzymes are involved in the transfer of electrons from cytosolic donors to FAD and then through 2 consecutive haem groups, to single electron acceptors on the opposing face of the membrane. The authors predicted that FRO2 is a trans-membrane
protein with 8 hydrophobic α-helices, 2 more than gp91phox. Four conservative histidine residues positioned in two trans-membrane α-helices probably coordinate the two haem groups located within the plasma membrane as it is predicted from the structure of gp91phox and the yeast ferric-reductases. The human gp91phox requires a second protein (p22phox) with 2 trans-membrane α-helices to form an active complex. In contrast, FRO2 seems to act alone. Even more, the C-terminal domain, including the two additional 2 trans-membrane α-helices shows similarity to p22phox. Ferric-chelate reductases from other species seem to be active also in plants. It was reported that the ectopic expression of yeast FRE1 and FRE2 (encoding Fe (III) reductases) in tobacco leads to overall increasing of the iron reductase activity along the entire plant. The FRE2 transformants were also more tolerant to Fe deficiency in hydroponics cultures as shown by higher chlorophyll and Fe concentrations in younger leaves (Samuelsen, 1998). Another element in the metal ion uptake system in plants is a group of transporters belonging to the ZIP family (for ZRT, IRT related Proteins) (Guerinot, 2000). IRT1 (Iron Regulated Transporter 1), an iron deficiency inducible protein, is the first isolated Arabidopsis ZIP protein (Eide et al., 1996). The function of IRT1 to transport Fe (II) is confirmed by functional expression in yeast. Additional studies in yeast showed that IRT1 has a broad substrate range and transports Mn2+ and Zn2+ and possibly Cd2+; on the other side, cadmium, copper, cobalt and Fe (III) inhibit it (Korshunova et al., 1999). Meanwhile several other metal transporters have been identified in A. thaliana - ZIP1-4, ZNT1, AtNramp1/3/4, COPT1 and LCT1, which makes obvious that multiple pathways of plant metal transport exist. (Clemens, 2000).
The third element of the Strategy I respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion by a root plasma membrane H+-ATPase. Strong increase in the plasma membrane H+-ATPase activity occurs under Fe-deficiency (Dell'Orto et al., 2000; Rabotti and Zocchi, 1994 ). By pumping protons outside the cell, this enzyme contributes to enhance the solubility of Fe oxides and generatesthe proton motive force for ion uptake. In addition, its activity would help to maintain an adequate environment for the activity of the Fe-deficiency-induced PM Fe(III)-chelate
reductase (low apoplastic pH and membrane potential homeostasis). Unlike Fe(III)-chelate reductase, an increased H+-ATPase activity was not frequently observed. The enzyme activity seems to differ considerably between plant species and genotypes (Bienfait, 1988; Buckhoutet al., 1989; Chosack et al., 1991; Rabotti and Zocchi, 1994;Schmidt et al., 1997). It has been suggested that Fe(III) reduction and proton extrusion activities may be regulated independently (Yi andGuerinot, 1996).
Physiological disorder in the tomato mutant chloronerva
The chloronerva is a spontaneous mutant of Lycopersicon esculentum Mill. Cv. Bonner Beste (BB). The mutant was extensively studied during the last decades and a lot of information concerning its physiology was collected. The mutant chloronerva is characterized by severe disturbance of iron metabolisms. It exhibits intercostal chlorosis of young leaves, retarded growth of root and shoots, excessive root branching and permanently activated iron uptake systems such as highly active ferric-chelate reductase, and enhanced proton extrusion. It shows also other typical iron deficiency syndromes as thickening of the root tips and increased density of the root hairs. In spite of these features, the mutant accumulates iron in roots and shoots (Scholz et al., 1988; Becker et al., 1995). Iron-phosphate particles are formed in the chloroplasts of palisade parenchyma of the leaves, and in cytoplasm and vacuoles of rhizodermis. At the electron microscopic level, it becomes obvious that the chlorotic leaves have a reduced number of chloroplasts (about one third in comparison with normal leaves). Moreover, the amount of the thylakoid membranes in the chloroplast is also reduced and disordered (Adler and Scholz, 1986). Besides iron, several other metals are accumulated in chloronerva – manganese and zinc in the leaves (Stephan and Grün, 1989) and copper in the roots (Pich and Scholz, 1991; Herbik et al., 1996). A similar process of metal accumulation is observed in the wild type BB under iron deficiency (Pich et al., 1994). Such an increased metal concentration and especially that of iron and copper could provoke formation of free radicals (Halliwell and Getteridge, 1984; Luna et al., 1994). This is in agreement with the observed 2-3 fold increasing of the activity of the catalases and
peroxidases in the roots of chloronerva, as well as in the wild type BB under iron deficiency (Pich and Scholz, 1993). Other stress-inducible proteins such as Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH, E.C. 1.2.1.12.) and Formiat-Dehydrogenase (FDH, E.C. 1.2.1.2.) are also up regulated (Herbik, 1997).
It was found that the chloronerva phenotype could be changed to wild type by grafting the mutant to BB rootstocks or by treatment with water extracts from normal plants. The chemical basis of this phenotypic normalization was shown to be a water-soluble, heat-stable, ninhydrin-positive substance, which was identified as non-proteinogenous amino acid nicotianamine (NA) (Scholz, Rudolph, 1968). NA is ubiquitous in the plant kingdom. The only known higher plant without NA remains the chloronerva mutant of BB.
The gene responsible for the chloronerva phenotype was fine-mapped onto the long arm of chromosome 1 and YAC clones surrounding the region were isolated using flanking markers (Ling et al., 1999). The chloronerva transcript was identified by cDNA isolation with the complementing cosmids. The gene encodes a novel protein of 35 kDa. The mutant differs from the wild type only by a single base pair change (T->C) at nucleic acid position 761 that creates a substitution of a phenylalanin by a serine at amino acid position 238.
Iron acquisition mechanisms of Strategy II
In contrast to all other higher plants, the economically most important Gramineae use a different iron uptake system known as Strategy II. The basic mechanism is excretion of phytosiderophores (PS), which act as chelators for ferric ions. The resulting Fe (III)-phytosiderophore complexes (Fe (III)-PS) are then taken up by root cells by a specific Fe (III)-PS transporter.
Most steps of the biosynthesis of PS have been deduced. The group of Satoshi Mori from University of Tokyo, Japan has investigated extensively the PS biochemical pathway and particularly that of mugineic acid family (MAs). The synthesis begins with methionine, which is synthesized in the roots but not transported from the leaves (Nakanishi et al., 1999). The methionine is adenosylated to S-adenosyl-methionin (SAM) by ATP-dependent SAM synthetase (SAMS). Three molecules of SAM are used to produce
nicotianamine by Nicotianamine synthase (NAS), perhaps the key enzyme in MA synthesis. In Strategy II plants, NA in addition to its function in Strategy I plants, is also a precursor of MA. The Nicotianamine aminotransferase (NAAT) catalyzes the transfer of an amino-group and subsequent reduction at 3’-carbon of the keto acid to 2'-deoxymugineic acid (DMA) (Higuchi et al., 1994). This crucial step distinguishes graminaceous plants from the other members of the plant kingdom. IDS3 and IDS2 (iron deficiency specific clones 3 and 2) (Nakanishi et al., 1997; Nakanishi et al., 2000) are the putative hydroxylase encoding genes that convert DMA to MA and 3-epihydroxy-2'-deoxymugineic acid (epiHDMA), and MA to 3-hydroxymugineic acid (HMA), as well as epiHDMA to 3-epihydroxymugineic acid (epiHMA). The authors suggest that IDS3 is an enzyme that hydroxylates the C-2' positions of DMA and epiHDMA, while IDS2 hydroxylates the C-3 positions of MA and DMA (Kobayashi et al., 2001). These phytosiderophores are produced in various ratios in the different graminaceous plants. It was found that the hydroxylated PS species possess enhanced chelate stability and affinity for iron (von Wirén et al., 2000). The iron-chlorosis resistance plants as barley and rye produce mainly hydroxylated PS while non-hydroxylated DMA predominates in the susceptible species such as maize and rice. (Kawai et al., 1988).
1.3. Nicotianamine and Nicotianamine synthase
Structure and possible functions of NAThe Strategy I and Strategy II appear rather different but they share a common element. In both strategies, the NA is a key metabolite. As it is known from the chloronerva mutant, the lack of NA leads to catastrophic disorder of the plant physiology due to the iron starvation and iron intoxication simultaneously. In Strategy II plants, one can predict similar or even stronger consequences of NA insufficiency since NA in these plants is also a precursor in PS biosynthesis.
NA was firstly isolated from Nicotiana tabacum leaves (Noma et al. 1971). Chemically NA is (2S:3’S:3’’S)N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidin-2-caboxyl acid (Kristensen and Larsen, 1974) (Fig.
1.1.). The biosynthesis of NA involves the direct condensation of three molecules of S-adenosyl-L-methionine followed by the formation of an acetidine ring (Mori and Nishizawa, 1987; Shojima et al., 1990; Kawai et al., 1988, 1990; Higuchi et al., 1994). The synthesis is catalyzed by nicotianamine synthase (NAS).
Fig. 1.1. Structural formula of Nicotianamine
NA is present in all higher plants. It is not present in the unicellular Algae and the bacteria. The only known NA-free multicellular plant is the tomato mutant chloronerva. Some Streptomyces strains as well some fungi (Basiolobus meristoporus) are also known to produce NA (Suzuki et al., 1996; Scholz et al., 1992)
Fig. 1.2. Three-dimensional structure of NA-Fe (II) complex
The concentration of NA in the plant tissues depends on the age of the tissue and the physiological stage (Rudolph et al., 1985). It varies from 10 to 400 nmol NA per gram fresh tissue, where the concentration of NA is highest
NH
N
NH
2in young developing tissues as root- and shoot epical meristeme (Stephan et al., 1990; Pich et al., 1994).
The 3-dimensional structure and the 6 functional groups of NA are optimal to form an octahedral complex with 2-valent metals (Fig. 1.2.) (Buděšinský et al., 1980; Ripperger and Schreiber, 1982).
The complex possesses an unusual kinetic stability, which prevents auto-oxidation of Fe(II) to Fe(III). Therefore, it was accepted for a long time that NA binds only Fe(II) but not Fe(III). However, it was demonstrated that NA is an effective chelator of Fe(III) at physiological pH values (von Wirén et al., 1999). Many authors have speculated about the putative function of NA, but this question remains to be clarified. NA has been proposed to play a key role in the cellular distribution of Fe (Scholz et al., 1992). The distribution of iron in apoplasm and symplasm of BB and chloronerva led to the conclusion that NA is not required for the transport of Fe(II) through the plasmalemma into the cell (Becker et al., 1992).
The function of NA is certainly related to its molecular structure since changes in the octahedral coordination structures in NA derivates destroys its capability to act as a “phenotypic normalization factor” of the chloronerva mutant (Scholz et al., 1988). Immunochemical detection in leaf and root cells localizes NA into cytoplasm around the vacuoles, except in the cells of the plants cultivated in a medium with high iron concentration (100 µM FeEDTA). In these plants, NA was located also within the vacuoles (Pich et al., 2001). Furthermore, an increased NA concentration has been reported due to increased Fe levels (Pich et al., 2001). The authors suggest the hypothesis that NA might play an important role in the detoxification of intracellular Fe and propose a function of NA as intracellular Fe-storage and buffering system.
Perhaps NA is a regulator of active iron keeping it available in solution and maintaining a small iron pool for all dependent cellular processes (Stephan and Scholz, 1993).
Nicotianamine synthase
The nicotianamine synthase (NAS, EC 2.5.1.43.) is the enzyme positioned on the metabolic crossroad of the Strategy I and Strategy II of iron acquisition.
The NAS synthesizes NA by direct condensation of three molecules of S-adenosyl-L-methionine followed by the formation of an acetidine ring (Kawai et al., 1988). The process could be reproduced in cell-free systems (Shojima et al., 1989).
NASs are a new class of enzymes, their number growing rapidly since the first cloning of the NAS from barley (Herbik et al., 1999; Higuchi et al., 1999) and the chloronerva gene of tomato (Ling et al., 1999). Today, at least 9 barley NAS genes are known, and at least 7 of them are unique genes.
The NASHOR1 and NASHOR2 genes (Herbik et al., 1999) were isolated from iron-deficient barley roots. This material was a suitable source for NAS isolation since the iron deficiency resulted in ~5 folds increasing ot NAS activity in the roots of barley (Herbik, 1997). After purification, the enzyme could be enriched about 140-fold. The resulting single protein peak precisely corresponded to the peak of enzyme activity. The SDS/PAGE of the purified active protein fraction revealed the presence of three polypeptides with molecular weights of 24, 28, 38 kDa. The 28-kDa protein was the only one, which could be labelled by UV cross linking to [14C]SAM, suggesting that this polypeptide binds SAM as it would be expected for NAS. Tryptic digestion of the 28-kDa polypeptide and micro-sequencing of the resulting peptides revealed several amino acid sequences that were used to generate oligonucleotide primers further used for 3’-RACE. A 0,6-kb DNA fragment was amplified from total RNA isolated from iron deficient barley roots. The 0,6-kb fragment was used to screen a phage library of barley cDNA clones. Several clones have been isolated. The nucleotide sequence of the full-length NASHOR1 clone (NCBI Accession No: AF136941) predicts a polypeptide of 330 amino acids residues and molecular mass of 35 611 Da. A full-length NASHOR2 (AF136942) clone predicts a 340 amino acids and a molecular mass of 36 312 Da. These two genes were cloned and the further analysis will be described in this work.
2. Materials and Methods
2.1. Materials
2.1.1. Plant materialPlant species Cultivars
Arabidopsis thaliana L. cv. “Columbia”
Hordeum vulgare L. cv. “Bonus“
Lycopersicon esculentum Mill. cv. “Moneymaker”
Lycopersicon esculentum Mill. cv. “Bonner Beste“
Lycopersicon esculentum Mill. chln mutant of cv. “Bonner Beste” Nicotiana tabaccum L. cv. “Petit Havana”
Tab. 2.1. Used plant species
2.1.2. Bacterial strains
Microorganism Strain Characteristics
Escherichia coli DH5α RecA1, endA1, gyrA96, thi-1, hsdR17, (rK-mK+), relA1,
supE44, u80∆lacZ∆M15, Tn10, (Tet)r, (Sambrook et al.,1989) Escherichia coli HMS174 (DE3) F -, recA, hsdR (rk 12-mk12+) RifR(DE3) Agrobacterium tumefaciens C58C1Rf r Deblaere et al. (1985)
2.1.3. Plasmids
Vectors Characteristics Source
pET12a ampr, T7 Novagen pET17b ampr, T7 Novagen
pBinAR19 Kanr, CaMV 35S Höfgens and Willmitzer, 1990
pAH17 Ubi Christensen and Quail, 1996
PCR2.1 Invitrogen
PCR2.1-TOPO Invitrogen
Tab. 2.3. Used plasmid vectors
2.1.3.1. Vector maps
Fig. 2.1. pBinAR19 vector used for dicots transformations. Contains expression cassette
based on cauliflower mosaic virus (CaMV) 35S promoter, partial pUC19 polylinker and octopine synthase terminator (OCS3). NPT II - neomycin phosphotranspherase from Tn5 (kanamycin resistance), LB and RB - respectively left and right border.
Fig. 2.2. pAHC17 vector used for monocots transformations. The vector is based on
maize Ubi-1 sequence. Ubi-1 elements: P - promoter sequence, E - exon, I - intron. NS3' - nopaline synthases 3' polyadenylated sequence. Arrow at the Ubi-1 exon indicates the transcription start site and the direction; pUC 8 - partial pUC 8 sequence. NS3` Hind III BamH I Xba I Sal I Pst I Sal I Pst I Xba I Xba I EcoR I Xba I P E I Xba I Pst I Sal I Hind III Sph I pUC 8 Hind III (768) 35S CaMV EcoR V (441) EcoR I (0) OCS3 BamH I (545) Xba I (551) Sal I (557) Pst I (567) Sph I (573) Sma I (542) Kpn I (536) RB LB NPT II
2.1.4. Primers and oligonucleotides
2.1.4.1. PCR primers
Name Notes Sequence 5'-3' 35S-P1 35S prom.
primer
ATG ACG CAC AAT CCC ACT ATC CTT C
AR1 NASARA1,
BamH I site
CCG CGG GAT CCA TGG GTT GCC AAG ACG AAC AAT TGG TGC AAA C
AR2 NASARA1,
BamH I site CCG CCG GAT CCC GTC CTC CTT AAG ACA ACT GTT CC
AR3 NASARA5,
BamH I site CCG CCG GAT CCA TGG CTT GCC AAA ACA ATC TCG TTG TG
AR4 NASARA5,
BamH I site CCG CCG GAT CCT ACT CGA TGG CAC TAA ACT CCT C
BC1 BCKDH,
Kpn I site AAC TTT AGC GGT ACC ACT AGG ACT TA
BC2 BCKDH,
Kpn I site ATC ACT TCG GTA CCG TTC GTG TAC AGG AGT GCT TAT BC3 BCKDH AAC TTT AGC TCA ACC ACT AGG ACT TA
BC4 BCKDH CAT TTG GAA AGA GAA CAT GAA GAT T BC5 BCKDH GTT TCA ATG GCA ACT TGG TTT TTA AG BC6 BCKDH GTG ACA TTA TGC TTG AAA ATC AGT AGG BC7 BCKDH TTC CCA TCG ACG CGA ATA CTT C
BC8 BCKDH TGG CCG TTT TCC AGT ATT CTA TTT C BC9-SphI BCKDH,
Sph I site ACA TGC ATG CGT TTC AAT GGC AAC TTG GTT TTT AAG BC10-SalI BCKDH,
Sal I site ACG CGT CGA CGT GAC ATT ATG CTT GAA AAT CAG TAG G BC11-SphI BCKDH,
Sph I site GTA TAT ATG CAT GCG TTT CAA TGG CAA CTT G BC12-SalI BCKDH,
Sal I site
TTT CTT TTC AGT CGA CGT GAC ATT ATG CTT G NASH1 (4-29) NASHOR1 GAT GCC CAG AAC AAG GAG GTT GAT GC
PCR PRIMERS CONTINUED... NASH1
(981-956) NASHOR1 CAC TTC CGC GTT GGT GAA CTC CTC TC M3USal I NASMET,
Sal I site CCT AAG GAG GTC GAC TAT GAG CTG CTA C M3UNde I NASMET,
Nde I site GGA GAA CCC ATA TGA GCT GCT ACA TCT AC M3RBamH I NASMET,
BamH I site GCG AGT GGG ATC CTT TAT CAT GGG AAG T
NA13 NASHOR1,
BamH I site CCG CCG GAT CCG ATG CCC AGA ACA AGG AGG TTG ATG C
NA14 NASHOR1,
Nde I site CCG CCA TAT GGA TGC CCA GAA CAA GGA GGT TGA TGC
NA15 NASHOR1,
Nde I site
CCG CCG GAT CCC AAC GAT CAG AAG GCC ACT TG
NA16 NASHOR1,
Kpn I site
CCG CCG GTA CCC GAT GCC CAG AAC AAG GAG GTT GAT GC
NA23 NASHOR2,
Nde I site
CCC GGC ATA TGG GCA TGG AGG GCT GCT GCA GCA AC
NA24 NASHOR2,
BamH I site CCC GGG ATC CGG CAT GGA GGG CTG CTG CAG CAA C
NA25 NASHOR2,
BamH I site CCC GGG GAT CCC TAC GCC TCC ATC TCC TCC ATG GCG AT
NA28 NASHOR2,
BamH I site ATC CCG GAT CCT GCA TGA GGA CAA ACG GAT TAT TAC C Tab. 2.4. Used PCR primers
2.1.4.2. Sequencing oligonucleotides
Name Note Sequence 5'-3'
AR9 5’ CY5,
NASARA1 AAA CCA GAA GAG AAG CGA GTG AGT AR10 5’ CY5,
NASARA1 AGA CAA CTG TTC CTC CCT AGC TCC AR11 5’ CY5,
NASARA5
TGC GTG TGA GTC GAT GTC AAA GTT AR12 5’ CY5,
NASARA5 TAC TCG ATG GCA CTA AAC TCC TC HPM1 5’ CY5,
HASHOR1 prom. GGA GCA GAG GCG GAT GAT HPM2 5’ CY5,
HASHOR1 prom. CAA AAT CAA ACG CCG GTT GTA AC HPM3 5’ CY5,
HASHOR1 prom. TGT GTG ATG CCT AGC CGT TCG HPM4 5’ CY5,
HASHOR1 prom. CAC GCA TGA TCT TCC AAC GAA TG HPM5 5’ CY5,
HASHOR1 prom. GTG TGG CGC CGA ACG CTT AG HPM6 5’ CY5,
HASHOR1 prom. ATC CCA CAT TAT GCC TAC CAT CAA AC HPM8 5’ CY5,
NASHOR1 prom.
TAA CCC TAA CCC CAT CAT ATC CCT AAC AC NAH1Pm-S1 NASHOR1 prom GG GCG AAG TGG AGT GGT GGA TG
NAH1-3’U1 NASHOR1 CGT TGC GAG GGA ATG AAA ATG AAG NAH1-3’R1 NASHOR1 TAC TTG GCA CAC TAC CCT CGT CTG
2.1.4.3. Oligonucleotides and DNA probes used for genetical mapping and detection of the barley NAS genes
Name Note Sequence 5'-3'
hvNAS-Uni-R NAS universal GCC AGC ATG TCG GAG TAG TGC GCC TC NASHOR1-F NASHOR1 CTG TGC TCT AGG TCG CCA CAA CAT ACA hvNAS2-F hvNAS2 CTC CTG TGC CTG TCC TGA GGT ACC AAG AA hvNAS3-F hvNAS3 CTA CTT CAC TCA CAC TAG TGC CCA GAA AGA AG hvNAS-Uni-F NAS universal ACG TCG TCT TCC TGG CCG CGC TC
hvNAS4-R hvNAS4 TAC ATA GGT GAT AGG TGG TGG TAG GAG GAG GAG TA
hvNAS5-R hvNAS CCC ATC AAT GTG CAG GGT ATC ATC TG hvNAS7-R hvNAS7 CAC ATT TCT TTT CCT TTG CAC AGT CTC TTG NAH1OP1 NASHOR1 TCT GGG CAT CCA TTT TAA TAC TGT ATG TTG NAH2OP1 NASHOR2 AGC TAA GCT GAG AGG CTG TGA GAG TGA GTG
Tab. 2.6. Oligonucleotides and DNA probes used for genetical mapping and detection of the barley NAS genes.
2.1.5. Enzymes and kits
Company Products
Amersham, Braunschweig Megaprime DNA labeling kit, Readyprime II DNA
labeling kit, Restriction endonucleases
Applied Biosystems, USA BigDye terminator sequencing kit Biolabs, USA Restriction endonucleases Biomol GmbH, Hamburg Total RNA isolation kit
Roche (Boehringer Mannheim) Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, Shrimp alkaline phosphatase, Klenow enzyme, Taq DNA polymerase, Expand high fidelity PCR system, Rapid DNA ligation kit, PCR nucleotide mix, Pwo DNA polymerase
Gibco-BRL, USA Superscript II RNase H- Reverse Transcriptase Invitek GmbH, Berlin Invisorb spin plant DNA extraction kit
Invitrogen, The Netherlands TA cloning kit, TA TOPO cloning kit
Novagen, USA His-Tag purification kit, T7-Taq purification kit. Qiagen, Hilden QIAquick agarose gel extraction kit, QIAqiuck PCR
purification kit, Plasmid purification kits (mini, midi and maxi), Taq PCR polymerase, Taq PCR master mix, DNeasy plant DNA isolation kit, DNeasy 96 plant kit, RNeasy plant total RNA isolation kit
Stratagene, Heidelberg Restriction endonucleases
USB, Cleveland OH, USA Restriction endonuclease, Klenow enzyme Tab. 2.7. Used enzymes and kits
2.1.6. Chemicals
Company Products
Sigma-Aldrich Adenosyl-L-methionine, Homocystein, S-Adenosylhomocystein, Methionine, Homoserin, Methylthioadenosin, Sodium carbonate, Sodium bicarbonate, Sodium chloride, Tween20, Potassium
chloride, Sodium monophosphate, Sodium diphosphate, IPTG, X-gal, MOPS
Ambion, USA RNAse ZAP cleaning reagent
Amersham, Braunschweig [α32P] dATP, [γ32P] ATP, [α32P] dCTP, Hybond-N+ nylon
membrane, S-Adenosyl-L-[carboxyl-14C]-methionine
Amresco, USA Phenol
Biometra, Göttingen Chloroform, Phenol, Phenol-chloroform, ATP, BSA, dNTPs, SDS
Difco, USA bacto-agar, bacto-trypton, yeast extract
Duchefa, The Netherlands Murashige-Skoog whole medium solid substance, Rifampicin, Kanamycin, Hygromycin, Carbenicillin
Fluka, Schweiz DEPC
Gibco-BRL, USA Agarose, 1Kb DNA ladder, EDTA
Invitrogen, The Netherlands RM basis medium, induction basis medium
Kodak, USA X-Ray films
Merck, Darmstadt Ethanol, Ethidium bromide, Formamide, HEPES, Magnesium chloride, Sodium acetate, Sodium hydroxide, Sodium-dihydrogen phosphate, di-Sodium hydrogenphosphate, trichloroacetate, Tris base Metabion, Planegg-Martinsried DNA oligonucleotides
MWG-Biotech AG, Ebersberg DNA oligonucleotides
NEN, USA GeneScreen Plus hybridization transfer membrane Roth, Karlsruhe Phenol, Phenol-chloroform, Chloroform, Formaldehyde,
Glycerol, Isopropanol, Lithium chloride, Sodium chloride Schleicher&Schuell, Dassel Blotting paper GB 002, nitrocellulose membrane BA 85 Serva, Heidelberg X-Gal, Sodium citrate, Tween20, tetracycline,
Coomassie blue, EDTA, X-gal Eurogentec, Belgium Smart Ladder
2.1.7. Laboratory tools and equipment
Company Equipment
AGS, Heidelberg DNA gel-electrophoresis tanks Appligene-Oncor Vacuum blotter
Berhof GmbH, Eningen DAP III high pressure block
BioRad, München Gene-Pulser, Mini Electrophoretic System (Mini-Protean SDS-PAGE running cell, Mini Trans-Blot Electrophoretic transfer cell, Electro Eluter)
Biotec Fischer, Reiskirchen Phero-stab 200 electrophoresis power supply CBS, USA EBS 250 power supply
DuPont, USA Sorvall centrifuge RC 5C
Eppendorf, Hamburg Mastercycler5330 (DNA- thermocycler), Thermomixer
5436 and 5437, Thermomixer compact, cold centrifuge 5402, BioPhotometer
GFL, Burgwedel Hybridization oven, water bath
Heraeus, Osterode Centrifuges (Biofuge 13, Biofuge 15R), HERASafe laminar boxes
Hofer, San Francisco CA,
USA Transfer electrophoresis unit
Millipore, Schwalbach Centricon protein concentrators, MilliQ water purification system
OWL Agarose gel trays
Perkin-Elmer, USA GenAmp PCR system 9700 (0,5 and 0,2 mL blocks) Pharmacia, Freiburg Photometer, Ultrospec plus
Polaroid, Offenbach MP-4 camera
Raytest, Straubenhardt FUJI BAS imager, imaging plates
Savant SpeedVac SPD101B
Stratagene, Heidelberg UV-Stratalinker 1800, NucTrap probe purification columns
Varian, Australia SpectAA 10 plus AAS Tab. 2.9. Used laboratory tools and equipment
2.1.8. Media 2.1.8.1. Bacterial media LB medium • 10,0 g NaCl • 5,0 g Tryptone, • 5,0 g yeast extract • H20 to 1000 mL pH 7,4 SOC medium • 0,580 g NaCl • 0,186 g KCl, • 20,0 g tryptone, • 5,0 g yeast extract,
• 2 mL of 2 M sterile (0,2 µm filter) glucose added after autoclaving • H2O to 1000 mL pH 7,4 TBY medim • 5,0 g NaCl • 5,0 g MgSO4.7H2O • 10,0 g tryptone • 5,0 g yeast extract • H2O to 1000 mL pH 7,4 YEB medium • 0,5 g MgSO4.7H2O • 5,0 g beef extract • 5,0 g peptone • 5,0 g saccharose • 1,0 g yeast extract • H2O to 1000 mL pH 7,0
2.1.8.2. Plant growth media
Self made ½ MS modified medium with variable iron concentration:
Stock solutions for self-made ½ MS medium (modified) (for 1 L stock solution):
• Stock 1 (40x): 3,61 g MgSO4 / 3,40 g KH2PO4 (or 7,38 g MgSO4.7H2O) • Stock 2 (100x): 82,50 g NH4NO3
• Stock 3 (100x): 95,00 g KNO3
• Stock 4 (100x): 16,60 g CaCl2 (or 21,98 g CaCl2.2H20) • Stock 5 (250x): 10 mM FeEDTA
For iron free medium, stock 5 was omitted.
Solutions of micro elements (1000x):
• 0,0125 g CoCl2.6H2O (or 15,30 g Co(NO3)2.6H2O) • 0,0125 g CuSO4.5H2O • 3,1000 g H3BO3 • 0,4150 g KI • 8,4500 g MnSO4.H2O • 0,1250 g Na2MoO4.2H2O (or 0,0927 g (NH4)6Mo7O24.4H2O) • 4,3000 g ZnSO4.7H2O •
To prepare ½ MS modified medium, Stocks 1 to 5 and the Microelements were added to 1x final concentration. The Stock 5 (iron source) was added in different volumes in order to obtain the desired iron concentration in the medium. The normal iron concentration for A. thaliana was 40 µM FeEDTA and pH was adjusted to 5,8 by 0,1 M KOH.
Hoagland liquid medium with variable iron concentration for tobacco, tomato and A. thaliana: Stocks • Stock 1 (100x) - 0,5 M Ca(NO3)2 • Stock 2 (100x) - 0,5 M MgSO4 • Stock 3 (100x) - 0,5 M KNO3 / 0,1 M KH2 PO4 • Stock 4 (1000x) - 10 mM FeEDTA Solutions of microelements • 5,0 mM H3BO3 • 4,5 mM MnCl2 • 3,8 mM ZnSO4 • 0,3 mM CuSO4 • 0,1 mM (NH4)6Mo7O24
To prepare 1 L of Hoagland medium, 10 mL each of the Stocks 1 to 3 and 1 mL Microelements were added. The Stock 4 (iron source) was added in different volumes in order to obtain the desired iron concentration in the medium. The normal iron concentration for A. thaliana was 40 µM FeEDTA and pH was adjusted to 6,0 by 0,1 M KOH. For tobacco and tomato, the normal concentration was 10 mM and pH 5,5.
2.1.9. Software
DNA and protein sequence data were processed using the program package Lasergene version 4 and 5 of DNASTAR Inc., USA and BLAST (Basic Local Alignment Search Tool), (Altschul et al., 1990).
The autoradiography images were analyzed by TINA 2.09 of Raytest Isotopenmeßgeräte GmbH, Germany)
2.2. Methods
2.2.1. Basic cloning methods and sequencing
The standard molecular cloning methods (e.g. restriction digestion, ligation, DNA and protein gel electrophoresis) were performed according to Sambrook et al., 1989.
The transformation of E. coli was performed using the heat-shock procedure (Cohen et al., 1972) or electroporation (Inoue et al., 1990).
Plasmid DNA extraction and purification was done by using the standard methods described in Sambrook et al., 1989, or by using Qiagen plasmid kit. PCR products were purified with QIAquick PCR purification kit (Qiagen).
TA based cloning was performed using the pCR2.1 or pCR2.1-TOPO vector systems (Invitrogene).
DNA fragments were isolated and purified from the agarose gel with the QIAquick kit (Qiagen).
DNA sequences were determined in the Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben by the dideoxynucleotide chain termination method (Sanger et al., 1977). DNA was detected using Fluorescence-labeled Primers by the A.L.F. Sequencer (Pharmacia LKB) and the Autoread Sequencing kit (Pharmacia). Sequences of longer DNA fragments were determined by primer walking. Alternatively, the BigDye terminator sequencing kit (Applied Biosystems) was used on ABIPrism (Applied Biosystems) sequencer.
2.2.2. Extraction of plant genomic DNA
The rapid plant DNA extraction, PCR grade, was carried out according to Edwards et al. (1991). The leaf tissue (~100 mg) was grinded in liquid nitrogen and then 400 µL of extraction buffer (200 mM Tris-HCl pH 8,0, 250 mM NaCl, 125 mM EDTA, 0,5% SDS) were added and the mixture was shaken for 1 min. The leaf suspension was centrifuged for 5 min at full speed and the supernatant transferred into a new tube containing 300 µL of
isopropanol. The DNA was collected by centrifugation for 10 min, washed twice with 70% ethanol, and resuspended in 100 µL of H2O.
High purity plant DNA was extracted and purified using the following protocol: 1. Grinding of 200-300 mg young leaves by mortar and pestle in liquid
nitrogen
2. Collecting of the powder into 15 mL tube
3. Adding of 3,0 mL Lysis buffer (10 mM Tris pH 8,0, 10 mM EDTA, 0,1 mM NaCl), 0,8 mL 10% SDS and 20 µL proteinase K
4. Incubation for 2 h at 56 °C and moderate shaking 5. Cooling to room temperature (RT)
6. Adding of 1 mL saturated NaCl and mixing by inversion of the tube for 15 sec
7. Centrifugation for 15 min at 4000 rpm, RT
8. Transferring the supernatant into new tube with 10 mL ethanol cooled to –20 °C
9. Collecting of the precipitated DNA with sterile glass rod and transferring into 1,5 mL tube containing 96% ethanol
10. Collecting the DNA by centrifugation and drying of the samples 11. Resuspending in 500 µL TE buffer
12. Purifying by Phenol/Chloroform extraction
13. Precipitating of the DNA by adding of 0,1 vol. 3M K-acetate, pH 5 and 1 mL 96% ethanol
14. Centrifugation to collect the DNA and removing the ethanol 15. Washing twice by 70% ethanol and drying
16. Resuspending in 100-200 µL Tris buffer, pH 8,0
Later, this method was replaced by the use of DNeasy Plant kit, DNeasy 96 plant kit (Quiagen) or by Invisorb spin plant DNA extraction kit (Invitek).
2.2.3. Extraction of genomic DNA from Methanobacterium
thermoautotrophicum
To extract DNA from M. thermoautotrophicum, the method of Bintrim et al., 1997 was used with minor modifications.
1. Collecting the cells from 3 mL liquid culture by centrifugation 2. Resuspending in 200 µL TE buffer
3. Sonication of the cells, 300 sec 4. Adding of lysosime (0,5 mg/mL)
5. Incubation for 30 min at 37 °C with shaking 6. Adding of proteinase K (2,0 mg/mL)
7. Incubation for 30 min at 37 °C
8. Adding of 200 µL buffer B (250 mM NaCl, 100 mM EDTA, 4% SDS) + 30 µL of 5M Guanidine isothiocyanate
9. Gently agitating by inverting the tube 10. Incubation at 68 °C for 1 h
11. Adding of 60 µL CTAB buffer (2% CTAB, 100 mM Tris-Cl pH 8,0, 20 mM EDTA, 1,4 M NaCl)
12. Incubation at 65 °C for 15 min
13. Phenol-chloroform extraction and isopropanol precipitation of the DNA 14. Dissolving the pellets in appropriate buffer
2.2.4. Southern blotting
DNA was prepared using the methods described above. For Southern hybridization DNA was digested with restriction enzymes, separated on a 0,5 to 1% agarose gel in Tris-acetate buffer (Sambrook et al., 1989) and
transferred onto a Hybond N+ (Amersham) or GeneScreen Plus (NEN)
membrane using alkali capillary blots according to the following protocol: 1. Pre-wetting of the membrane in distilled water for a few seconds 2. Equilibrating of the membrane in 0,4 N NaOH for 10-15 min 3. Agitating the gel in 0,25 N HCl for approximately 10 min
5. Transferring of the DNA to the membrane by using 0,4 N NaOH as a transfer solution on a capillary blot or vacuum blotter (Appligene-Oncor).
6. UV cross-linking the DNA to the membrane by using the Auto Cross link mode of UV Stratalinker 1800 (Stratagene).
2.2.5. Extraction of plant total RNA
The protocol for RNA isolation is based on the Guanidium thiocyanate (GCN) method of Chomzynski and Saccini, 1987 (modified):
Solution D:
• 250 g (GCN)
• 293 mL H2O (DEPS treated)
• 17,6 mL 0,75 M Na Sarkosyl (sodium lauryl sarcosinate)
• Mix and store at 4 °C. Before use, add 72 µL β-mercaptoethanol to 10 mL solution D.
DEPS H2O (RNAse free)
1 mL DEPS to 1 L H2O, 12-24 h at 37°C with shaking. Autoclave until the smell of DEPS disappeared (2-3 times).
Procedure:
1. Homogenization of the sample in 300 µL Solution D 2. Adding of 30 µL 2M Sodium Acetate, pH 4, vortex 3. Adding of 300 µL Phenol (pH 6,6), vortex
4. Adding of 60 µL Chloroform:Isoamyl alcohol (24:1), intensive shaking for 10 sec
5. Incubation on ice for 15 min
6. Centrifugation, 10 000 g, 20 min at 4 °C
7. Transferring of the aqueous phase to clean Eppendorf tube containing 300 µL Isopropanol
8. Incubation for 1 h at -20 °C
10. Aspirating the isopropanol
11. Washing twice in 70% ethanol and drying 12. Dissolving in H2O (DEPS treated)
13. Determination of the concentration and A260/A280 ratio.
Alternatively, total RNA isolation reagent (Biomol) or RNeasy kits (Qiagen) were used.
All the glass- and plastic ware used for RNA isolation were treated with RNAse ZAP cleaning reagent (Ambion) and washed with DEPS treated water.
2.2.6. Northern blotting
Formaldehyde-agarose (FA) gel electrophoresis preparation:
10x formaldehyde gel running buffer (FA gel buffer)
• 40 g MOPS (200 mM) in 800 mL 50mM Na-Acetate, pH 7,0 (with 2N NaOH)
• 20 mL 0,5 M EDTA, pH 8,0 • Adjusting to 1000 mL
• Autoclaving at 120 °C (2 bar) for 10 min.
1X FA gel running buffer (1 L)
• 100 mL 10X FA gel buffer
• 20 mL 37% formaldehyde (works without formaldehyde too) • 880 mL RNase free water
Gel preparation - 100 mL 1% agarose
• 1,0 g agarose
• 10 mL 10x buffer MOPS buffer • RNase-free water to 100 mL • Melting and cooling to 65°C • 1,8 mL Formaldehyde (37%) • 1 µL Ethidium bromide (10 mg/mL)
5x RNA loading buffer (1 mL)
• 1,6 µL Saturated bromphenol blue • 8,0 µL 0,5 M EDTA, pH 8,0 • 72,0 µL 37% Formaldehyde • 200,0 µL 100% Glycerol • 308,4 µL Formamide • 400,0 µL 10X FA gel buffer • RNase-free water to 1 mL 20xSSPE • 3,00 M NaCl • 0,20 M NaH2PO4 • 0,02 M EDTA Adjusting the pH to 7,4 Sample preparation
• Add 1 volume of 5X RNA loading buffer per 4 vol. RNA • 3-5 min at 65°C, keep on ice until loading into the gel.
Transfer of the RNA to the membrane
RNA was prepared using the methods described above. For Northern hybridization the RNA was separated on a 1% FA agarose gel and transferred onto a Hybond N+ (Amersham) or GeneScreen Plus (NEN) membrane using alkali capillary blots according to the following protocol:
1. Pre-wetting of the membrane in distilled water for a few seconds 2. Equilibrating of the membrane in 10xSSPE for 15 min
3. Soaking the gel in 5 volumes distilled water for about 5 min to remove the formaldehyde from the gel, repeat 4 times
4. Transferring of the RNA to the membrane by using 10xSSPE as a transfer solution on a capillary blot or vacuum blotter (Appligene-Oncor)
5. UV cross-linking the RNA to the membrane by using the Auto Cross link mode of UV Stratalinker 1800 (Stratagene).
2.2.7. Radioactive labeling of DNA probes Oligonucleotide labeling by terminal phosphorylation
• 100 ng oligonucleotide in less than10 µL volume • 2,5 µL 10X polynucleotide kinase buffer
• 5,0 µL [γ-32P] ATP (50 µCi) • H20 to 25 µL
• 1 µL T4 polynucleotide kinase (10 U/µL), vortex gently • Incubate 1 h at 37°C
• Reaction was stopped by adding of 75 µL STE buffer (100 mM NaCl, 20 mM Tris/HCl pH 7,5, 10mM EDTA)
• Purification by NucTrap probe purification columns (Stratagene).
Labeling of long DNA probes by random priming
• 25 ng DNA probe
• 5 µL Random nonamer primers mix • H2O to 50 µL
• Denaturation - 5 min, 100°C • Span down, kept on RT
• 10 µL Labeling buffer (dATP, dGTP, dTTP in Tris/HCl pH7,5, 2-mercaptoethanol and MgCl2)
• 5 µL α-32P dCTP (50 µCi) • 2 µL Klenow (1 U/µL) • Incubation 10-30 min, 37°C
• Denaturation for 5 min at 100°C and chilling on ice for use in hybridization or the reaction was stoped by addition of 5 µL 0,2 M EDTA
Alternatively, Megaprime or Readyprime II labeling systems (Amersham) were used.
2.2.8. DNA-DNA and DNA-RNA hybridization protocol
Hybridization of the Southern and Northern blot membranes was carried using the method of Church (Church and Gilbert, 1984) (modified).
Church buffer 250 mL • 4,40 g NaH2PO4 • 16,55 g Na2HPO4.2H20 • 17,50 g SDS • 2,50 g BSA • H20 to 250 mL, dissolve overnight
• Filter before use to remove the remaining SDS crystals.
Phosphate buffer, 100 mL • 74,7 mL 0,5M Na2HPO4 • 25,3 mL 0,5M NaH2PO4 Wash buffer • 80 mL Phosphate buffer • 50 mL 20% SDS • 4 mL 0,5 M EDTA (pH 8,0) • H2O to 1 000 mL Prehybridization
The membranes were prehybridized at 55-65 °C with Church buffer containing denaturated carrier DNA (Calf thymus DNA) with concentration of 100 µg/mL.
Hybridization and washing the membrane
New preheated Church buffer, denaturized carrier DNA and the labeled DNA probe were added to the membrane. Hybridization was carried overnight at the same temperature as the prehybridization. Then, the membrane was washed with the wash buffer (1x30 min, 4x15 min) and the signal was
detected and quantified with a Bio-Imaging analyzer BSA2000 (Fuji Photo Film Co. Ltd) or X-ray film.
Stripping of the membranes
After the hybridization, the membranes must not dry out. To strip the labeled probe, a boiled 0,5% SDS solution was poured onto the membranes and allowed to cool to room temperature.
2.2.9. Protein overexpression, isolation and purification
The protein overexpression in E. coli was carried according to standard methods (Sambrook et al., 1989).
Purification and solubilisation of inclusion bodies
The induced cells were harvested by centrifugation and resuspended with appropriate buffer. In case of NAS overexpression, a buffer A (50 mM Tris, 1 mM EDTA, 3 mM DTT, 500 µM Methionine, pH 8,7) was used. Then the E. coli cells were destroyed by ultrasonication and the suspension was centrifuged at 10 000 g, 4 °C. The supernatant contained the soluble proteins while the natant contained the inclusion bodies and insoluble cell debris. Typically, the NAS protein was found in the inclusion bodies fraction.
Refolding of the inclusion bodies.
The following protocol for purification and refolding of NAS was used (for pellets obtained from 500 mL LB culture):
1. Washing the pellets with 0,1 M Tris pH 8,0
2. Resuspending of the pellets in 15 mL buffer (0,1 M Tris pH 8,0, 1mM MgCl2, 0,2 mg/mL Lysozym, 1000 U/mL Benzoat)
3. Incubation for 1 h at 37 °C
4. Centrifugation at 10 000 g, 4 °C for 15 min
5. Washing of the pellets twice with buffer A and once with H20
6. Resuspending of the pellets in 20 mL 2M Urea by shaking for 20 min at RT
8. Dissolving of the pellets in 80-100 mL 6M Urea by shaking at RT (the protein concentration must not exceed 1 mg/mL)
9. Centrifugation at 10 000 g, RT, for 15 min
10. Dialyzing the suspension against 50 fold higher volume of Urea in buffer A with decreasing concentration of the Urea (5 M, 4 M, 3 M, 2 M, 1 M, 0M (twice)), at 4 °C for several days
11. Centrifugation at 10 000 g, 4 °C, for 20 min. Refolded NAS protein was controlled by SDS- PAGE.
A further preconcentration of the proteins (when needed) was performed by using Centricon concentrators (Millipore).
2.2.10. Antibody preparation and purification
Rabbit was injected with 80 µg of purified NAS protein for 4 times with an interval of 1 month in between. Two weeks after the 3rd injection a serum sample was taken to check the polyclonal specificity. Two weeks after the 4th injection the animal was killed and its blood was collected. The polyclonal serum was obtained by centrifugation of the collected blood and the polyclonal IgG fraction was isolated using protein A sepharose (Amersham). Further, the IgG graction of the serum was purified according to following protocol:
1. Resuspending of about 1 mL of drained gel in 50 mL PBS buffer pH 7,0 and degassing for 10 min
2. Packaging of the gel slurry in C10/10 column (Amersham) 3. Washing of the gel with 30 mL of PBS buffer
4. Adding of 3 mg of serum protein to the surface of the column and allowing it to penetrate through the column by adding a buffer
5. Waiting for 30 min at RT to complete the binding process between IgG and protein A.
6. Washing of the column with PBS until the outlet buffer has no protein content, as measured with OD at 280 nm
7. Elution of IgG by using 0,1 M of glycine buffer pH 3,0
8. Collecting of the outlet in 3 mL collection tubes and immediately neutralization using a saturated solution of KOH
9. Precipitation of the IgG protein with 50% ammonium sulphate 10. Collecting the protein by centrifugation
11. Reconstition of the pellet in PBS pH 7,0 and 12. Dialyzing in the PBS buffer
13. Measuring of the protein content and dispensing and storing of the polyclonal IgG.
2.2.11. Western blot analysis
Soluble proteins were separated in a denaturing 12% (w/v) SDS-PAGE gel. After electrophoresis, the proteins were transferred in transfer buffer (25 mM Tris, 192 mM glycine pH 8,3, 20% v/v methanol) to the nitrocellulose membrane (Schleicher and Schuell) at 1,3 mA/cm2 for 16 h on Mini Trans-Blot Electrophoretic transfer cell (Bio-Rad) or on a bigger device.
Immunodetection of the blotted NAS proteins
To detect the blotted NAS protein, Alkaline-Phosphatase bound anti-rabbit antibodies were used according the following protocol:
1. Blocking of the excess protein binding sites of the membrane by incubation in 3% BSA in TBST buffer (10 mM Tris pH 8,0, 150 mM NaCl, 0,1% Tween20) for 30 min at RT
2. Incubation of the membrane with specific NAS antibodies diluted 1:10 000 in TBST for 1 h
3. Washing of the membrane by gently shaking in TBST, 3x5 min, RT 4. Incubation with second antibodies (Anti-rabbit Ab,
Alkaline-Phosphatase conjugate, Boehringen) diluted 1:10 000 in TBST for 1 h 5. Washing of the membrane by gently shaking in TBST, 3x5 min, RT 6. Developing the color as follows: adding of 90 µL NBT (75 mg/mL nitro
blue tetrazolium) and 70 µL BCIP (50 mg/mL 5-bromo-4chloro-3-indolyl phosphate) (both from Novagen) per 20 mL AP buffer (100 mM Tris, pH 9,5, 100 mM MgCl2) (Novagen) and incubation of the membrane at RT until color develops
2.2.12. Screening of the barley BAC library
Filters containing spotted barley BAC library (cv. Morex, constructed in Clemson University, USA, provided by D. Schmidt, IPK) were hybridized with specific oligonucleotide probes (NAH1OP1) by using the following protocol:
Prehybridization solution and conditions:
• 6x SSPE
• 5x Denhardt’s reagent (see below)
• 0,5% SDS
• 100 µg/mL Calf thymus DNA
Denhard’s reagent (100x):
• 2% (w/v) BSA, • 2% (w/v) Ficoll,
• 2% (w/v) PVP (polyvinylpyrrolidone)
Prehybridization was carried out at 55 °C, overnight.
Hybridization solution and conditions:
• 6x SSPE
• 5x Denhard’s reagent
• 0,5% SDS
• 100 µg/mLL Calf timus DNA
• 200 ng 5’-labeled (γ-32P ATP) oligonucleotide probe Hybridization at 55°C, overnight.
Washing solution and conditions:
First wash (in the hybridization tube)
• 2xSSPE
• 0,1% SDS
Second wash (in tray)
• 2xSSPE
• 0,1% SDS
Washing for 30 min at 55 °C
Third wash (in tray)
• 2xSSPE
• 0,1% SDS
Washing twice for 30 min at 55 °C
Fourth wash (in tray)
• 0,5 SSPE
• 0,1% SDS
Washing twice for 30 min at 55 °C
The signals were detected by exposing the membranes on X-ray films and amplification screens, for 4 days at -80 °C.
2.2.13. Agrobacterium tumefacience growth and treatment Transformation of Agrobacterium tumefacience
The competent cells of Agrobacterium tumefacience (pGV 2260) were prepared using the CaCl2 method. The Agrobacterium strain was grown in 50 mL of YEP medium at 28 °C until OD600~ 0,5-1,0. The cells were centrifuged at 3000 rpm for 5 min and resuspended in 1 mL of 20 mM of CaCl2. 100 µL aliquots of the resuspended cells were dispensed in separate Eppendorf tubes. The transformation with plant expression vectors was done using the thawing-freezing method as described by Höfgen and Willmitzer, 1988.
Total DNA preparation from Agrobacterium tumefacience
The bacteria were grown in 5 mL YEP at 28 °C for 18 hours and the pellets were collected by centrifugation in a 1,5 mL tube. After resuspension in 300 µL of suspension buffer (20 mM EDTA, 50 mM Tris pH 8,0), 100 µL of 5% sarkosyl in TE and 2,5 mg/mL proteinase K in TE were added. The mixture
was incubated at 37 °C for 2 hours, followed by two extractions with phenol, one with phenol-chloroform (1:1) and finally twice with chloroform. The supernatant was precipitated by adding 0,1 volume of 3 M K-Acetate and 2 volumes of ethanol, the DNA pellets were washed twice with 70 % ethanol and resuspended in 50µL TE buffer.
2.2.14. Plant growth and treatment 2.2.14.1. Arabidopsis thaliana
Growth of A. thaliana in soil
Arabidopsis plants were grown in a growth chamber at 22°C with 6000 lux of white light for 16 hours. The plants grew at these conditions until the end of maturation (~22 days after pollination).
Hydroponics cultures of A. thaliana
Using hydroponics methods in air- and light-conditioned growth chamber by using Hoagland medium with 40 µM FeEDTA performed the growth of A. thaliana under controlled conditions.
The plants were grown in 30x24 cm trays, as it is described in Fig. 2.3. The plants were grown in a chamber with 200-300 µE/cm2 light intensity, 8 h light by 21 °C and 16 h darkness at 19 °C (short day). The seeds were germinated on rock wool bundles that were soaked into the nutrient solution. At day 3-4 the seedlings had 2 cotyledons developed and the opaque screen was removed to permit the light to the plantlets. At day 8, the nutrient solution was changed and the glass plate was shifted to allow the insertion of aeration tubes. At day 10-11 the plantlets usually were well developed (2-6 leaves), the glass plate was completely removed, and the growth tray was taken out of the germination chamber. The plants were grown further with regular changing of the nutrition medium until the day 32-36 when they were used in physiological experiments in iron limited conditions.
Fig. 2.3. Hydroponics system for A. thaliana. Figure (A). The germination chamber: A
small tray (5), covered with hole-plate (1) with a rock-wool bundle soaked into the nutrient solution (2) in each hole. The whole construction (1, 2, 5) was put into a bigger tray (6), covered with a glass plate (3) and opaque screen (4). The A.
thaliana seeds were put on the top of each rock-wool bundle. Wed paper towels on
the bottom of the big tray kept the humidity in the germination camera. After the germination, the opaque screen was removed to allow lighting of the seedlings.
Figure (B). The growth tray. After approximately 8 days, the glass plate was shifted
at few centimeters and a tube for aeration (7) of the nutrition medium was inserted. After 2 weeks approximately, the seedlings were well developed. Then the small tray was taken out of the germination camera and the plants were grown until the end of the experiment by regular changing of the nutrition medium.
Submerged A. thaliana cultures
For short-term iron limitation experiments (up to 1 week of iron limitation) and to prepare a material for screening of DNA arrays, submerged A. thaliana cultures were used.
1. About 10 surface-sterilized (Ethanol, NaOCl) A. thaliana seeds in 100 mL flasks, containing 25 mL ½ Murashige-Skoog (MS) modified medium
2. Imbibition of the seeds for 24 h at 4 °C in the dark
3. Moving the flasks in a growth chamber and growing for 10 day at constant shaking
4. Removing of the nutrition medium and washing of the plants with 0,1 mM Ca(NO3)2 for 30 sec, 0,1 mM Ca(NO3)2/1 mM EDTA for 10 min and finally again in 0,1 mM Ca(NO3)2 for 30 sec
5. Transferring of the plants in new iron-free (10% HCl washed) 100 mL flasks filled with 25 mL ½ MS modified medium with or without iron.
Plant transformation of Arabidopsis thaliana by vacuum infiltration
Transformation of Arabidopsis was performed based on the protocol of Bechtold et al. (1993). Plants of Arabidopsis thaliana (ecotype Columbia) were grown for three weeks under short day conditions (8 hours light, 16 hours dark) and transferred to long day (16 hours light, 8 hours dark). After three weeks, the emerging bolts were cut to induce growth of multiple secondary bolts. Vacuum infiltration of plants with A. tumefacience culture was done one week after clipping. Bacteria were grown until OD600 > 2,0, harvested by centrifugation and resuspended in three volumes of infiltration medium (OD600 approx. 0,8). Entire shoots of the plants were submerged into the A. tumefacience suspension in a beaker. Vacuum was applied by an oil pump for 5 min and then rapidly released. Plants were removed from the beaker, placed on their side and kept at high humidity under plastic warp for 24 hours, after that they were uncovered and set upright. Seeds were harvested from the siliques, sterilized by Na-hypochlorite as described before and plated onto GM selection plates containing 50 mg/L hygromycin. After two weeks, hygromycin resistant plants were transferred to soil, grown up and their seeds were collected. Stable transformation and expression of the constructs were analyzed by PCR.
2.2.14.2. Nicotiana tabacum growth and treatment
Transformation of tobacco and tomato
The leaf disk method was used for Agrobacterium-mediated transformation of tobacco and tomato as it is described in Horsch et al. (1985)
Hydroponics cultures of tobacco
The regenerated from leaf disks transgenic tobacco was grown in hole-plate covered trays with liquid Hoagland medium with 10 µM FeEDTA as a normal iron concentration. When seeds were used, they were surface-sterilized and germinated on MS agar plates where they grew for 2 weeks, and then the plantlets were transferred to trays with Hoagland medium.
2.2.14.3. Lycopersicon esculentum growth and treatment
The tomatoes were transformed and grown in a way similar to tobacco except the germination of the seeds, which was performed on wet paper towels. Than the seedling were transferred to Biolaston (PVC fibers) soaked in diluted 1:1 with water Hoagland medium as is described on Fig. 2.4..
Fig. 2.4. Grow system for tomato seedlings. A tray was filled with Biolaston (1) soaked in
nutrient solution. The seeds were put on the top of a plastic mesh (2). Constant level of the nutrient solution was maintained by the system (3).
2.2.15. Nicotianamine synthase activity assay
Preparation of the samples for determination of NAS activity in plant extracts was performed according to A. Herbik (1997).
The activity of the NAS enzyme was determined by using S-Adenosyl-L- [carboxyl-14C]-methionine (14C-SAM) as a substrate. 50 µL plant or bacterial extract was incubated with 20 µM 14C-SAM at 30 °C, pH 8,7 for 10 min. Adding an equal amount of 99% methanol stopped the reaction. The samples were centrifuged and 0,5 to 2 µL of the supernatant was spotted on silicagel plates together with 5µL 1 mM NA solution and separated by thin layer chromatography (TLC) by using 1-propanol/water in 7:8 ratios as a mobile phase. To visualize the non-labeled NA on the TLC-plate, the latter was dried and stained by ninhydrin (300 mg Ninhydrin, 3 mL Acatic acid, 100 mL butanol) at 60 °C. The amount of the produced 14C NA was quantified by using Fuji Bio-Imaging analyzer and the Software TINA 2.09.
2.2.16. Determination of the metal ions concentration
The determination of the concentration of iron, zinc and other metal ions was performed according to Stephan et al. (1994). Absolutely dry plant material was dissolved in 65% HNO3 at 170°C for 3 h in High pressure block DAB III (Berhof GmbH) and the samples were measured on the Atomic absorption spectrometer SpectAA 10 Plus (Varian).
2.2.17. Determination of the Nicotianamine concentration
For determination of the NA concentration plant material was crushed in liquid nitrogen. The powder was mixed with water and the resulting pull was homogenized in a Potter-Elvehjem device after thawing. The homogenate was stirred for 1 h and deproteinized by heating to 80 °C and centrifuged at 48 000 g for 20 min. The supernatant was concentrated in a vacuum evaporator at 42 °C and lyophilized. The lyophilizate was dissolved in 0,2 M Na-citrate buffer pH 3,05. The NA concentrations were determined after postcolumn derivatization with ninhydrin at 570 nm using an amino acid analyzer (S 432, Sykam) as described in Schmidke and Stephan (1995).
2.2.18. Ferric-reductase assay
Ferric-reductase activity was used as a criterion for the response of the plant to iron insufficiency. The measurement of Fe3+-chelate reduction by intact
roots was followed by observing the formation of Fe2+-BPDS (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid) complex from Fe3+-EDTA, according to Chaney et al. (1972). Plants with appropriate size were transferred into 100 mL aerated opaque glass tubes filled with Hoagland medium containing 2 µM FeEDTA. The plants were grown in this condition for 5 days. During this time, the pH of the medium was measured regularly each day. At the 5th day the roots of the plants were washed in 10-4 M Ca(NO3)2 solution and the plants were transferred to new tubes filed with iron-free Hoagland medium with pH adjusted to the last pH value measured in the previous nutrient solution. Then, 1 mL 2 mM FeEDTA and 1 mL 8,5 mM BPDS were added to the medium. The first sample (T0 – the base) was taken after 30 sec waiting for mixing then samples after 30 min (T1) and 60 min (T2) incubation time were also collected. All the samples were stored at dark until the spectrophotometric measurement of the extinction at 540 nm. At the end of the experiment, the exact volume of the reaction medium was measured in order to be used in the calculations. The iron reductase activity was estimated according the rule that 1 M Fe(BPDS)3 has an extinction of 22500 units at 540 nm and the result was recalculated for the reaction volume and the time to obtain the activity in µmol reduced Fe3+ per hour per liter (µmol/h/L).
To control the induction of the iron-uptake mechanisms in A. thaliana grown to produce a material used in the screening of DNA arrays for iron-concentration dependent gene expression, a slightly modified procedure was used.
2.2.19. Chlorophyll content measurement
The samples were grinded in mortar and liquid nitrogen. Then 1 mL 100% acetone was added and the samples were extracted for 10 min with shaking. The homogenates were centrifuged for 6 min at 10000 g at 4°C. The supernatant was transferred to a new tube and the debris was extracted again with 80% acetone (10 min, vortex). The second extract was added to the first one. The A652 of the supernatant was measured against 80% acetone. The samples were diluted with 80% acetone where was necessary. The A652 value was divided with 34,5 to obtain the chlorophyll concentration in mg/mL. The quantity of pigment was normalized to the sample weight in each sample
3. Results
Iron deficiency leads to in a significant increase in NAS activity in roots of barley – a typical strategy I plant. Therefore, barley roots grown under iron deficient conditions have been chosen as a starting material for NAS isolation. A SAM-binding protein was detected in the protein extract and was used for microsequencing. Based on the amino acid sequence a specific oligonucleotide was derived and used as probe for the screening of a barley cDNA library. Two cDNA clones designated as NASHOR1 and NASHOR2 were isolated. Further studies were mainly focused on the NASHOR1 gene, which was used for expression studies and generation of transgenic plants. By homology search, two genes similar to NASHOR1 sequences were identified in the A. thaliana genome on chromosome 1 and chromosome 5 and were designated as NASARA1 and NASARA5, respectively. These NAS-like sequences were used for plant transformations.
3.1. Arabidopsis thaliana wild type experiments
Wild type Arabidopsis thaliana was studied in order to obtain basic physiological data concerning NA and metal metabolism in this plant.
Arabidopsis plants were grown in nutrient solution with different iron concentrations. An increase in NA was observed both in the shoots and in the roots of plants grown in iron limiting condition (0 µM and 1 µM FeEDTA) whereas in the normally iron supplied (40 µM EDTA) or iron overloaded (100 µM FeEDTA) plants the NA concentration was considerably lower (Fig. 3.1.)
Shoot NA [nmol/g (FM)] 0 100 200 300 400 500 0 µM Fe 1 µM Fe 40 µM Fe 100 µM Fe Root NA [nmol/g (FM)] 0 100 200 300 400 500 0 µM Fe 1 µM Fe 40 µM Fe 100 µM Fe
Fig. 3.1. Nicotianamine concentrations (nmol/g FW) in shoots and roots of A. thaliana plants grown in different iron concentration of the medium. The bars represent
the standard error.
The iron concentrations were maximal in the normal iron supplied plants (Fig. 3.2.). A slight decreasing of the iron in the plant was observed in iron-overloaded plants perhaps due to an inhibitory effect of the high iron concentration in the nutrient solution. The iron in the iron-limited plants was considerably lower. This tendency was clearer in the shoots than in the roots of the plants.
